Since the introduction of biological and chemical weapons during the First World War, intensive research and development by the super power nations has led to the creation of large stockpiles of chemical weapons and new technologies for the delivery of these weapons. These weapons of mass destruction are so named because of their ability to kill enormous numbers of people in a short period. Weapons of this nature are attractive to both developing countries and terrorist groups because they are easier and cheaper to acquire than nuclear weapons. Moreover, the technologies and synthetic methodologies for manufacturing some chemical weapons are openly available and easily accessible to the public. Terrorists may seek to obtain greater status or bargaining power against their more developed enemies by demonstrating that they have the technological capabilities required to develop, produce, and deliver chemical and biological warfare agents.
Although the use of biological and chemical weapons is banned by international treaty, these weapons are thought to be in the stockpiles of several extremist nations and terrorist organizations. The release of the nerve gas sarin in the Tokyo subway system in 1995, killing 12 and wounding over 1000, demonstrated the consequences of chemical warfare technology in the hands of terrorists and/or anarchists. In the United States, the threat and fear of potential terrorist attacks using biological and chemical weapons has been particularly elevated since the attacks of Sep. 11, 2001.
Nerve gases, such as sarin, soman, tabun, and VX, are classified chemically as organophosphate compounds. Organophosphates are characterized as stable, easily dispersed, and highly toxic, with toxicity taking effect rapidly both when absorbed through the skin and via respiration. The threat of organophosphate poisoning is not limited to exposure to nerve gases, as commercial pesticides such as malathion and parathion are also organophosphate compounds that are toxic upon exposure. In the United States, approximately 20,000 reported organophosphate exposures occur per year; however, it is estimated that only 1% of field worker illness from pesticide exposure is reported. Internationally, organophosphate poisoning occurs in virtually every country in the world. The United Nations reports that over 30,000 organophosphate-related fatalities occur worldwide each year. Third world countries have less legislation regarding safe agricultural use of pesticides; therefore, a much higher incidence of poisoning exists among field workers and the public who buy produce from these fields.
Organophosphate compounds inactivate cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), by phosphorylating the active site serine hydroxyl group on the enzyme, leading to the loss of ability to hydrolyze the substrate acetylcholine. AChE is the enzyme that terminates neurotransmission at the neuromuscular synapse. The normal function of cholinergic synapses requires that the neurotransmitter acetylcholine (ACh) be hydrolyzed by cholinesterases within several milliseconds to terminate neurotransmission. Organophosphate poisoning occurs when the inactivated enzyme is unable to break down ACh, leading to ACh accumulation throughout the autonomic nervous system, the somatic nervous system, and the brain, resulting in overstimulation of the acetylcholine receptors. Prolongation of neurotransmission results in extensive damage to the target cells and, frequently, death of the organism. Decreasing muscle strength leading to paralysis occurs when motor plates remain depolarized by persisting levels of acetylcholine. The inhibition of AChE by nerve gases generally results in death by asphyxiation within a few minutes, as control is lost over respiratory muscles.
AChE is highly concentrated at sites of nerve-muscle contact where it is attached to the specialized basal lamina juxtaposed between the nerve terminal and the postsynaptic membrane (reviewed in Massoulié et al., 1993; Legay, 2000; Rotundo, 2003). The only known function of AChE at the synapse is to rapidly hydrolyze the neurotransmitter acetylcholine thus terminating neurotransmission. The high concentration of AChE between the nerve terminal and the muscle membrane is maintained primarily by the muscle, and the newly synthesized AChE molecules are released into the synaptic cleft.
All AChE forms in vertebrates are encoded by a single gene and, in mammals, give rise by alternative splicing to one to three polypeptide chains of about 480 amino acids depending on the species. Birds appear to express only one variant, of higher apparent kDa, whereas most mammals express at least two alternatively spliced forms. All forms share the same catalytic domain containing about 95% of the total sequence, however the H form (AChEh) has a 30-40 amino acid carboxyl terminus that is cleaved post-translationally and a glycophosphoinositol (GPI) anchor covalently attached, whereas the T form (AChEt) that has a different 40 amino acid terminus that allows assembly with one of two non-catalytic subunits specifying subcellular localization (FIG. 1). The GPI-anchored form is expressed only in hematopoietic and lymphatopoietic tissues where its function is unknown. In addition to the catalytic subunits, there are two non-catalytic subunits encoded by separate genes that associate with the enzyme to target it to specific regions of the cell surface, the collagenic tail (ColQ) and the transmembrane anchoring “p” peptide or PRIMA (reviewed in Massoulié et al. 1993; Legay, 2000; Rotundo, 2003).
Only the AChEt form is expressed in nerves and muscle in most vertebrates. The major forms of AChE expressed in neurons are monomers and dimers and in particular the tetrameric form covalently linked to the small transmembrane PRIMA peptide that anchors it to the plasma membrane. The minor forms are intracellular or secreted and appear to be a precursor pool in the secretory pathway, the endoplasmic reticulum and the Golgi apparatus. In skeletal muscle, the major forms are the soluble globular and collagen-tailed versions of AChE with little or no expression of other variants (FIG. 1). The most important form is the collagen-tailed AChE that is the predominant, if not unique, form at the neuromuscular synapse. The inactivation of this form at the neuromuscular junction of the diaphragm is usually the proximal cause of death.
The appearance of collagen-tailed AChE forms is dependent upon expression of the collagenic tail itself, ColQ (FIG. 2). The ColQ is encoded by a separate gene and is expressed in many tissues including skeletal muscle. The ColQ molecule is composed of several distinct functional domains including the N-terminal domain (NTD) that associates covalently via SH bonds with the catalytic subunits, the triple-helical collagenic domain, and the C-terminal domain (CTD) responsible for anchoring the ColQ AChE to the synaptic basal lamina in skeletal muscle. Within the N-terminal domain is the 17 amino acid PRAD sequence, the Proline-Rich Attachment Domain (Bon et al., 1997). Analysis of the N-terminal domain showed that this region is responsible for the covalent attachment of the catalytic subunits to the ColQ (Bon and Massoulié, 1997; Bon et al., 1997), and moreover, that co-expression in COS cells with the catalytic subunit resulted in increased formation of tetramers from dimers (Bon et al., 1997). Similar observations were made using the mouse muscle C2/C12 cell line (Legay et al., 1999). In fact, the PRAD peptide can even induce assembly of the tetrameric AChE from dimers in solution (Chitlaru et al., 2001). A model for the molecular interactions responsible for this association based on the crystal structure of the complex has been presented recently (Dvir et al., 2004). Similar to ColQ, the transmembrane anchoring “p” peptide (PRIMA) also comprises a PRAD sequence within its N-terminal domain that can induce tetramerization of AChE.
Current strategies for treatment of individuals exposed to organophosphate compounds include reactivation of inactivated AChE using oxime reactivators, prophylactic administration of muscarinic antagonists such as atropine, and placement of the victim on a ventilator if necessary. Oxime reactivators such as 2-pyridine aldoxime methiodide (2-PAM) restore the function of inactivated AChE by displacing the covalently bound organophosphate molecule from the inactivated enzyme. However, poisoning by some nerve agents, such as soman, is complicated by the inhibited enzyme going through an “aging” process (Strayer reaction) that renders it incapable of being reactivated by any oxime. Atropine binds to muscarinic acetylcholine receptors to protect against excess acetylcholine-mediated neurotransmission resulting from AChE inhibition. However, atropine treatment has no direct effects on the inactivated AChE, the nerve gas, or on nicotinic acetylcholine receptors. In cases of severe nerve gas poisoning, large doses of atropine need to be taken until the level of functional AChE is restored. Moreover, in spite of ongoing developments in these types of treatments, the fatality rate could remain as high as 35% with large-scale exposure during a military conflict.
Another strategy employed to reduce the deadly effects of nerve gas exposure is to pretreat individuals at risk of exposure to organophosphate compounds with active site antagonists such as pyridostygmine bromide (PB). However, this strategy has its own harmful drawbacks. PB is a carbamate compound that is thought to protect AChE by reversibly binding to (“carbamylating”) it, so that the nerve agent cannot bind to it. It may also assist in protection against nerve agent by “desensitizing” ACh receptors. However, PB treatment may lead to bromide intoxication from prolonged consumption of excessive doses of bromide, causing protean symptoms, particularly psychiatric, cognitive, neurological, and dermatologic (and some believe this may be the cause of the “Gulf War Syndrome”).
The most current research efforts to reduce the effects of exposure to nerve agents that inhibit AChE focus on the development of scavenging enzymes that stoichiometrically inactivate the nerve agent, or catalytic scavenging enzymes capable of hydrolyzing nerve agents in situ, in both cases reducing the effectiveness of the nerve agent. To this end, various forms of recombinant AChE, butyrylcholinesterase, paraoxonase, and other enzymes have been developed and studied for their effectiveness (Broomfield et al., 1991; Allon et al., 1998; Billeck et al., 1999; Broomfield et al., 1999; Saxena et al., 1997; 1998). However, at best these molecules would be administered systemically and would inactivate unreacted organophosphates but leave untouched the inactivated AChE molecules. Thus there still remains little that can be done for victims that have been exposed to high levels of organophosphates.
Conventional methods of treatment for victims of nerve agent or pesticide poisoning are thus limited to controlling the damage caused by nerve agent exposure and/or limited in effectiveness only with certain organophosphate compounds. Moreover, the costs associated with such treatments are not limited to the financial costs required to deliver massive doses of the drugs in cases of severe poisoning but also include the costs to individuals suffering from deleterious side effects resulting from treatment.
There is a need for a method of treating organophosphate poisoning that directly increases active AChE molecules. There is also a need for a more cost effective and efficacious treatment for nerve agent exposure. The invention is directed to these and other important ends.